Mass spectrometry is a technology which is widely used in most scientific disciplines that require accurate and precise measurement of elemental and molecular components. Its use in the pharmaceutical sector is often associated with the Drug Discovery and Development process. The primordial step to analyze a sample is to perform a sample treatment to enable its study. It really requires know-how to obtain good quality results. The following tips and tricks can help you to rapidly select a protein purification strategy and to anticipate some possible problems.
How are mRNA vaccines changing drug discovery?
mRNA vaccines are developing very quickly. There are already 25 ongoing clinical trials against cancers, as well as clinical trials against infectious diseases such as HIV. Recombinant monoclonal antibody (mAB) based treatments that have lead to the most successful therapies could even switch to mRNA coding heavy and light chains for direct antibody production into the patient (as of yet still at the level of mouse models).
Indeed, messenger RNAs (mRNA) are now new biological entities to take in consideration for the development of efficient and personalized therapies. They have several advantages over conventional drugs: [Read more…]
How can you improve your reporter gene assays?
Gene reporter assays are widely used in research and Drug Discovery. The development of such cell lines is often time-consuming and costly, sometimes upwards of 5-20k € per cell line. And once these cell lines are finally validated, why jeopardize your results with cheap and low-end detection kits? They could impact the reproducibility of your experiments in unintended and unanticipated ways. BPS Bioscience has developed two reliable and extremely powerful luciferase detection kits: the ONE-Step™ Luciferase Assay System (#60690) and the Dual Luciferase Assay System (#60683). [Read more…]
HyNic-4FB chemistry serving antibody-drug conjugates (ADCs)
Challenges and limitations of ADC development
One of the major challenges of modern medicine is to find a safe cancer therapy. The Food and Drug Administration has approved Adcetris® (brentuximab vedotin) and Kadcyla® (ado-trastuzumab emtansine) that are considered as powerful treatments against cancer. They are good examples of antibody-drug conjugates (ADCs). The principle is to link a drug, that can be cytotoxic, to an antibody able to target cancer cells. Unfortunately, the development of ADCs faces some difficulties. Indeed, depending on the method for the conjugation, the ADC can be highly heterogenous. [Read more…]
Hit-and-run cell-based therapy research with Trilink mRNA
This summer, Moffett et al. published in Nature Communications (Nature Communications – doi:10.1038/s41467-017-00505-8) a new approach for cancer therapeutic research and development based on mRNA delivery. They called it ‘Hit-and-run programming’ because of the simplicity of the method. Interestingly, the optimization of the delivery was performed with eGFP mRNA. This post describes how TriLink’s mRNAs contribute to the simple and efficient delivery of mRNA into cells.
ELISA test to confirm elimination of Host Cell Proteins (HCP) from bioproductions
Expression systems – removing their contaminants
CHO, E. Coli and Pichia are useful expression systems for drug production. This can be for proteins, enzymes or antibody productions. Research and development for therapeutic perspectives requires a high level of quality controls. Such expression systems contain +1000 host cell proteins (HCPs). A major challenge in biological component production is to remove this HCP contamination. In order to monitor removal efficiency (during protein purification, as mentioned further on in this article), Canopy Biosciences have developed and optimised HCP detection kits based on the principle of the sandwich ELISA test as illustrated below.
Principle of the HCP Detection assay is based on sandwich ELISA test – by Canopy Biosciences at tebu-bio.com
cAMP signals visualized in live human pancreatic primary cells
In a recent publication, researchers from the University of Miami Miller School of Medicine (USA) describe that Serotonin released by human beta cell inhibits glucagon secretion by alpha cells. They demonstrated that this paracrine loop was mediated via the cAMP pathway. To do so, they captured in live human pancreatic islet cells cAMP signals using a specific fluorescent biosensor.
Screen for inhibitors of PD-1 signalling with a complete cellular assay system
Immunotherapy represents a field in Drug Discovery which is quickly developing and leading to significant progress in treatments of a number of diseases, especially cancer. The approach is based on inducing, enhancing, or surpressing an immune response. Therapeutic manipulation of immunopathways has led to promising clinical results . The first therapeutic antibodies directed against the checkpoint receptor PD-1 have been already brought to the market (Nivolumab, Pembrolizumab) by Bristol Myers Squibb and Merck/MSD respectively, and approved for the treatment of diverse cancer types.
Today, I would like to review tools to build up a comprehensive assay set up for cell based inhibitor screening on PD-1 / PD-L1/PD-L2 binding. [Read more…]
Measure Antibody-Dependent Cell-mediated Cytotoxicity (ADCC)
Fig 1: Antibody-dependent cell-mediated cytotoxicity (ADCC)
ADCC is a simple but important mechanism for the immune system to target diseased or infected cells. Antibodies bind to specific antigens on the surface of the target cell (see Fig 1). PBMCs or natural killer (NK) cells, express Fc receptors on their cell surface and act as the effector cells. Interaction between the Fc region of the antibody and the Fc receptor induces the effector cell to degranulate, releasing IFN-γ, granzymes, and other cytotoxic compounds that lyse the target cell.
ADCC is not only a natural part of the adaptive immune response, but animal experiments have shown that it can also be seen as an important mechanism of action of therapeutic monoclonal antibodies (1), including the breast cancer drug trastuzumab, and rituximab, a drug used to treat diseases which show overactive, dysfunctional, or excessive numbers of B cells (e.g. lymphomas).
Cell lines to build up cellular ADCC screening assays
Fig 2: Principle of BPS’s ADCC cell lines
To enable researchers to build up a cellular ADCC screening system, BPS Biosciences have developed 2 reporter cell lines, which can replace NK cells or PBMCs in such a cellular assay (see Fig 2). The system is based on Jurkat cells that stably express human FcγRIIIa (CD16a), the receptor for the Fc region of human IgG. The FcγRIIIa on the Jurkat cells binds to the IgG on the surface of the target cell. This crosslinking causes the Jurkat cells to activate NFAT transcription, which induces the expression of luciferase and can be easily detected using the ONE-Step™ Luciferase Detection Reagents.
The effectiveness of ADCC depends on how well the effector cells are activated after the engagement of FcγRIIIa. Human FcγRIIIa displays dimorphism at amino acid 158 – one allele (V158) encodes a high Fc affinity receptor variant, while the other (F158) encodes a lower Fc affinity receptor variant. BPS offers 2 different ADCC cell lines expressing either of these Fc receptors to allow selective antibody binding analyses using each type of receptor.
- ADCC Bioassay Effector Cell, F variant (Low Affinity)
- ADCC Bioassay Effector Cell V variant (High Affinity)
Get more information about our ADCC cell lines – just leave your questions or comments in the form below!
Reference:
(1) Clynes, RA, Towers, TL, Presta, LG, Ravetch, JV; Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets; Nat Med. 6 (4): 443-446 (2000)
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Cancer Immunotherapy: Targeting CD73 to inhibit adenosine generation
Fig 1: CD39 and CD73 leading to elevated levels of extracellular adenosine.
Extracellular adenosine 5′-triphosphate (ATP) is released by dying and damaged cells, and it acts on many immune cells to promote inflammation. On the other hand, the unphosphorylated
metabolite, adenosine, functions as an anti-inflammatory molecule. Two extracellular ecto-5´-Nucleotidases, CD39, and CD73, convert extracellular ATP to ADP/AMP and AMP to adenosine, respectively, leading to elevated levels of extracellular adenosine (Fig 1). [Read more…]